Non-invasive mapping of cellular or subcellular events in living organisms, or molecular imaging, is an evolving and largely unexplored field. Magnetic resonance imaging (MRI) is used for in vivo cellular imaging and requires the use of cellular contrast agents. Many of the current developments in contrast agents have revolved around SPIO (super paramagnetic iron oxide) particles. These are specifically formed crystals of a mixture of ferrous and ferric oxides (magnetite). For MRI contrasting, these particles must be as small as possible and yet retain permanent magnetic properties (Bulte et al., 1994). Various companies have developed biologically compatible nanosphere and microsphere SPIO particles. These may be coated with a number of materials including protein, phospholipids, polysaccharide, dextran (Arbab et al., 2003) and silane polymer shells that may or may not further include targeting antibodies. However, they are not useful for long-term studies in which labelled cells divide and the SPIO contrast agent becomes diluted. In addition, SPIO particles alone cannot provide information on cellular and molecular function.
Gene expression systems are under development to couple protein expression with the formation of suitable contrast agents. Adenocarcinoma cells have been transfected with a ferritin gene cloned into a viral expression vector so that the cells will over-express heavy and light ferritin subunits (Genove et al., 2005). Upon culture in iron supplemented media, elevated cellular levels of ferritin provide a sufficient quantity of the crystalline ferrihydrite core for MRI.
Magnetosomes are endogeneous contrast agents that are membrane-bound structures produced by magnetotactic bacteria and contain magnetite or iron oxide mixtures (Bazylinski and Frankel, 2004). Due to their size specificity and distinctive crystal morphology, magnetosomes are good in vivo imaging contrast agents. The full complement of genes responsible for magnetosome synthesis in bacteria is still under investigation (Schüler, et al., 2006; Komeili et al., 2006; Grunberg et al., 2001). Reproducing the entire magnetosome structure in foreign cells has therefore not been accomplished. Current research and development has focussed on the isolation and characterization of the bacterial magnetosome particle (Schuler et al., Max Planck Institute for Marine Biology, Biannual Report, 2004-2006; Herborn et al., 2003).
U.S. Pat. No. 5,861,285 describes a method to make magnetic particles using transformed magnetic bacterium. U.S. Pat. No. 6,033,878 describes a fusion DNA sequence of a protein gene fused to a fragment of a magA gene coding for a protein bound to an organic membrane for covering magnetic particles produced in magnetic bacterium. U.S. Pat. No. 6,251,365 describes a magnetosome surrounded by a phospholipid membrane made by a formation process. U.S. patent application serial no. 2004/0048289 describes a protein specific to a magnetic particle membrane derived from a magnetic bacterium (Magnetospirillum sp.) AMB-1.
Magnetite has been reported in the tissue of numerous vertebrates; however, some mollusks and bacteria precipitate this iron mineral. It is the iron crystal that magnetotactic bacteria use to orient themselves in the earth's magnetic field. Crystalline magnetite has not been identified in mammals, nor has a definitive role been demonstrated for magnetosome-like structures in migratory birds. Hence, the role of magnetosomes in vertebrates, including mammals, is still speculative (Kirschvink et al., 2001).
An iron regulated gene, magA, which encodes a membrane-bound, iron-transport protein involved in magnetite synthesis has been identified in Magnetospirillum species of bacteria (Bazylinski and Frankel, 2004; Matsunaga et al., 1992; U.S. Pat. No. 6,033,878).
While the prior art has demonstrated that magnetic particles as contrast agents can be made in prokaryotic systems, there still remains a need to develop a method for developing contrast agents in eukaryotic cells for a variety of clinical diagnostic and therapeutic applications.